To be successful pathogens, bacteria require mechanisms for sensing the host environment, processing environmental changes, and making appropriate adaptations. In many bacteria, expression of disparate virulence factors is coordinately controlled by a common regulatory system. Virulence expression by Bacillus anthracis, the causative agent of anthrax, represents a unique example of a regulatory response to an environmental signal. The known virulence factors of B. anthracis, a tripartite exotoxin and a D-glutamyl polypeptide capsule, are produced in vitro only during growth under elevated levels of carbon dioxide in media containing bicarbonate. The effect of bicarbonate on toxin and capsule production is specific, and not simply due to its buffering capacity during bacterial growth. This CO2/bicarbonate signal is of physiological significance for a pathogen which invades mammalian host tissues. the long-term goal of this project is to elucidate the molecular basis for virulence gene expression in B. anthracis. Toxin and capsule synthesis may be co-regulated by trans-acting factors in response to the CO2/bicarbonate signal. Experiments described here will probe the regulation of pag, the gene for the immunogenic toxin protein, protective antigen. Transcription of this gene increases 10 to 40-fold in response to bicarbonate. B. anthracis plasmid pX01 is required for stimulation of pag transcription. We will identify trans-acting regulatory genes that affect expression of pag by using transposon-mediated insertional mutagenesis and by screening a pX01 library for clones which activate a pag reporter gene. A regulatory locus at least 3 kb upstream from pag on pX01 has already been implicated as necessary for protective antigen synthesis. This locus, and other regulatory loci identified in experiments proposed here, will be characterized by defining structural limits, sequencing, identifying open reading frames, and constructing null mutants. Effects of the CO2/bicarbonate signal on regulatory gene function and expression will be examined. The pag promoter will be analyzed in detail: upstream sequences important for CO2-dependent transcription will be identified. These experiments will elucidate components of the B. anthracis signal- transduction system responsible for recognition and transmission of the CO2/bicarbonate signal and possibly global control of virulence factor synthesis by this organism. Results of this study will enhance awareness of host-parasite relationships and signal transduction and provide information relevant to the pathogenesis of anthrax disease.